Comprehensive Multiphase (CMP) NMR Monitoring of the Structural Changes and Molecular Flux Within a Growing Seed.

A relatively recent technique termed comprehensive multiphase (CMP) NMR spectroscopy was used to investigate the growth and associated metabolomic changes of 13C-labeled wheat seeds and germinated seedlings. CMP-NMR enables the study of all phases in intact samples (i.e., liquid, gel-like, semisolid, and solid), by combining all required electronics into a single NMR probe, and can be used for investigating biological processes such as seed germination. All components, from the most liquid-like (i.e., dissolved metabolites) to the most rigid or solid-like (seed coat) were monitored in situ over 4 days. A wide range of metabolites were identified, and after 96 h of germination, the number of metabolites in the mobile phase more than doubled in comparison to 0 h (dry seed). This work represents the first application of CMP-NMR to follow biological processes in plants.

Comprehensive multiphase NMR: a promising technology to study plants in their native state.

Nuclear magnetic resonance (NMR) spectroscopy is arguably one the most powerful tools to study the interactions and molecular structure within plants. Traditionally, however, NMR has developed as two separate fields, one dealing with liquids and the other dealing with solids. Plants in their native state contain components that are soluble, swollen, and true solids. Here, a new form of NMR spectroscopy, developed in 2012, termed comprehensive multiphase (CMP)-NMR is applied for plant analysis. The technology composes all aspects of solution, gel, and solid-state NMR into a single NMR probe such that all components in all phases in native unaltered samples can be studied and differentiated in situ. The technology is evaluated using wild-type Arabidopsis thaliana and the cellulose-deficient mutant ectopic lignification1 (eli1) as examples. Using CMP-NMR to study intact samples eliminated the bias introduced by extraction methods and enabled the acquisition of a more complete structural and metabolic profile; thus, CMP-NMR revealed molecular differences between wild type (WT) and eli1 that could be overlooked by conventional methods. Methanol, fatty acids and/or lipids, glutamine, phenylalanine, starch, and nucleic acids were more abundant in eli1 than in WT. Pentaglycine was present in A. thaliana seedlings and more abundant in eli1 than in WT.

Comprehensive multiphase NMR spectroscopy of intact ¹³C-labeled seeds.

Seeds are complex entities composed of liquids, gels, and solids. NMR spectroscopy is a powerful tool for studying molecular structure but has evolved into two fields, solution and solid state. Comprehensive multiphase (CMP) NMR spectroscopy is capable of liquid-, gel-, and solid-state experiments for studying intact samples where all organic components are studied and differentiated in situ. Herein, intact (13)C-labeled seeds were studied by a variety of 1D/2D (1)H/(13)C experiments. In the mobile phase, an assortment of metabolites in a single (13)C-labeled wheat seed were identified; the gel phase was dominated by triacylglycerides; the semisolid phase was composed largely of carbohydrate biopolymers, and the solid phase was greatly influenced by starchy endosperm signals. Subsequently, the seeds were compared and relative similarities and differences between seed types discussed. This study represents the first application of CMP-NMR to food chemistry and demonstrates its general utility and feasibility for studying intact heterogeneous samples.

Mass spectrometric analysis reveals remnants of host-pathogen molecular interactions at the starch granule surface in wheat endosperm.

The starch granules of wheat seed are solar energy-driven deposits of fixed carbon and, as such, present themselves as targets of pathogen attack. The seed's array of antimicrobial proteins, peptides, and small molecules comprises a molecular defense against penetrating pathogens. In turn, pathogens exhibit an arsenal of enzymes to facilitate the degradation of the host's endosperm. In this context, the starch granule surface is a relatively unexplored domain in which unique molecular barriers may be deployed to defend against and inhibit the late stages of infection. Therefore, it was compelling to explore the starch granule surface in mature wheat seed, which revealed evidence of host-pathogen molecular interactions that may have occurred during grain development. In this study, starch granules from the soft wheat Triticum aestivum cv. AC Andrew and hard wheat T. turgidum durum were isolated and water washed 20 times, and their surface proteins were digested in situ with trypsin. The peptides liberated into the supernatant and the peptides remaining at the starch granule surface were separately examined. In this way, we demonstrated that the identified proteins have a strong affinity for the starch granule surface. Proteins with known antimicrobial activity were identified, as well as several proteins from the plant pathogens Agrobacterium tumefaciens, Pectobacterium carotovorum, Fusarium graminearum, Magnaporthe grisea, Xanthomonas axonopodis, and X. oryzae. Although most of these peptides corresponded to uncharacterized hypothetical proteins of fungal pathogens, several peptide fragments were identical to cytosolic and membrane proteins of specific microbial pathogens. During development and maturation, wheat seed appeared to have resisted infection and lysed the pathogens where, upon desiccation, the molecular evidence remained fixed at the starch granule surface.